CN103396995B - A kind of three-dimensional culture method screening breast carcinoma stem cell - Google Patents
A kind of three-dimensional culture method screening breast carcinoma stem cell Download PDFInfo
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- CN103396995B CN103396995B CN201310299224.2A CN201310299224A CN103396995B CN 103396995 B CN103396995 B CN 103396995B CN 201310299224 A CN201310299224 A CN 201310299224A CN 103396995 B CN103396995 B CN 103396995B
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Abstract
The invention discloses a kind of three-dimensional culture method screening breast carcinoma stem cell, adopt hydrogel to carry out dimensional culture to human breast cancer cell, directly obtain breast carcinoma stem cell microballoon, thus obtained microsphere cell is ESA
+cD44
+/ CD24
-/lowphenotype, obviously has the characteristic of tumor stem cell, and this inventive method is reliable breast carcinoma stem cell screening method.The present invention is simple to operate, and compared with the prior art such as flow cytometer sieve method, greatly reduce reagent consumptive material, instrument uses and the cost of the aspects such as manpower.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of three-dimensional culture method screening breast carcinoma stem cell.
Background technology
Mammary cancer is that one has a strong impact on one of even life-threatening modal malignant tumour of women's physical and mental health.In China, the morbidity of mammary cancer is obvious ascendant trend, has become the modal malignant tumour of women at present.The existence of tumor stem cell is the root place of malignant tumour to have scholar to think.Therapy study display for many years, the behaviors such as radiation of resisting in the resistance of breast carcinoma stem cell in radiotherapy and medicine elimination ability, chemotherapy process and postoperative recurrence and transfer to be still in its treatment comparatively stubborn problem.Therefore, need badly and develop suitable screening method and go out breast carcinoma stem cell with Effective selection, thus in order to the research of the aspects such as pathogenesis of breast carcinoma mechanism and biotherapy.
Prior art mainly adopts the method separating mammary tumor stem cell of selected by flow cytometry apoptosis or immunological magnetic bead sorting, and its cost is higher, and flow process is loaded down with trivial details, and obtain cell viability poor, follow-up cultivation and study difficulty large.
Research finds, tumor stem cell has the feature of some uniqueness, and this forms the population of cells come in every shape in contributing to cultivating in vitro.Sub-fraction cell as human glioma cell system U251 can form compact, circular group in cultivation, most cells in these groups all has the ability of self, tumour ball can be formed, and neurone, astroglia cell and oligodendrocyte can be divided into.Breast carcinoma stem cell has multiple subgroup, and scholars infer that it also has unique constructional feature.In cultivating in vitro, the population of cells that these characteristics perhaps can make breast carcinoma stem cell come in every shape.(the Ponti D such as Ponti, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res, 2005,65:5506-5511.) research discovery, from 3 routine breast cancer tissues and a breast cancer cell line determined, take out cell carry out vitro culture, the tumour cell ball that can not be sticked, in these cell balls, some cell has the undifferentiated cell of self and continuous multiplication capacity.This research is pointed out, and this in-vitro culture model can be used as one of method obtaining breast carcinoma stem cell.
Three-dimensional cell culture technology is by cell seeding in certain extracellular matrix, and extracellular matrix serves as growth support, cell can be broken up and produce certain three-dimensional tissue's specificity structure.In the branch field of the developmental biology such as organization formation, vascular development and Organ Reconstruction, Three-dimensional cell culture technology is widely used.Much research finds, the culture environment of external three-dimensional is more conducive to maintenance and the performance of cell function than the culture environment of two dimension.Research shows, Three-dimensional cell culture system can promote the activity of hemopoietic stem cell effectively, hemopoietic stem cell long-term surviving still can be kept to increase cell quantity, strengthen cell division capacity when the acellular factor exists.Consider biologic bracket material---collagen, in the application in stem cell in vitro large scale culturing field, is cultivated if breast cancer cell is seeded in the three-dimensional stent material be made up of collagen, will become a more effective breast carcinoma stem cell training method.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of three-dimensional culture method screening breast carcinoma stem cell is provided.
The technical solution used in the present invention is:
Screen a three-dimensional culture method for breast carcinoma stem cell, comprise the steps:
1) obtain the human breast cancer cell of unicellular, add in perfect medium, make cell suspension, cell density is adjusted to 1 × 10
4~ 1 × 10
5individual/mL;
2) salmon zymoplasm is added cell suspension, the concentration making zymoplasm is 1 ~ 4U/mL, and mix, obtain mixed liquor A, ice bath is for subsequent use;
3) with perfect medium, salmon procollagen is diluted to 1.5 ~ 3mg/mL, obtain mixed liquid B, ice bath is for subsequent use;
4) mixed liquor A and mixed liquid B equal-volume are mixed, obtain mixed solution C, be seeded to Tissue Culture Plate, be incubated at 36 ~ 38 DEG C, make it be frozen into soft gel;
5) above soft gel, add the perfect medium that volume is mixed solution C1 ~ 2 times, be placed in cell culture incubator and cultivate, every 2 ~ 4 days replaced medium once;
6) be cultured to 9 ~ 11 days, namely obtain breast carcinoma stem cell microballoon.
Preferably, the human breast cancer cell used in step 1) is MCF-7.
Preferably, described perfect medium is the DMEM in high glucose substratum being added with 10 ~ 15v/v% foetal calf serum, 80 ~ 150U/ml penicillin and 80 ~ 150mg/ml Streptomycin sulphate.
Preferably, step 2) in, in mixed liquor A, the concentration of salmon zymoplasm is 4U/mL.
Preferably, in step 3), in mixed liquid B, the fibrinogenic concentration of salmon is 2mg/mL.
Preferably, in step 5), culture condition is: 37.0 ~ 37.5 DEG C, 4.9 ~ 5.1%CO
2, saturated humidity.
The invention has the beneficial effects as follows:
The present invention adopts hydrogel to carry out dimensional culture to human breast cancer cell, and directly obtain breast carcinoma stem cell microballoon, thus obtained microsphere cell is ESA
+cD44
+/ CD24
-/lowphenotype, obviously has the characteristic of tumor stem cell, and therefore this inventive method is reliable breast carcinoma stem cell screening method.The present invention is simple to operate, and compared with the prior art such as flow cytometer sieve method, greatly reduce reagent consumptive material, instrument uses and the cost of the aspects such as manpower.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 cultivates the tumor stem cell microballoon formed.
Fig. 2 is the result of Immunofluorescence test CD44, CD24.
Fig. 3 is the result that real-time fluorescence quantitative PCR detects ESA, CD44, CD24 expression amount.
Fig. 4 is the result that Western-blot detects CD44, CD24 expressing quantity.
Embodiment
Studies have reported that, the physical strength of culture medium can regulate differentiation and the self of mouse endothelial stem cell.Softer matrix can maintain the self of endothelial stem cell, and harder matrix then can promote the differentiation of endothelial stem cell.The present invention makes the hardness of the hydrogel of formation within the scope of 80 ~ 100pa by regulating fibrinogenic concentration, breast carcinoma stem cell is bred in matrix, and maintains the characteristic of its self, thus screening obtains tumor stem cell microballoon.
Content of the present invention is set forth further below in conjunction with embodiment.In embodiment, MCF-7 cell used is provided by Chinese Academy of Sciences's Shanghai cell bank, and salmon Fibrinogen and salmon zymoplasm are all purchased from Reagent Protein company.
embodiment 1
1) by tryptic digestion MCF-7 clone, obtain the MCF-7 cell of unicellular, add in perfect medium (being added with the DMEM in high glucose substratum of 15v/v% foetal calf serum, 100U/ml penicillin and 100mg/ml Streptomycin sulphate), make breast cancer cell suspension, with perfect medium (being added with the DMEM in high glucose substratum of 10v/v% foetal calf serum, 80U/ml penicillin and 80mg/ml Streptomycin sulphate), cell density is adjusted to 10000 cell/mL;
2) salmon zymoplasm (0.1U/ μ L) 10 μ L are mixed with 250 μ L cell suspensions, obtain mixed liquor A, be placed in for subsequent use on ice;
3) with perfect medium, salmon Fibrinogen is diluted to 2mg/mL, obtains mixed liquid B, be placed in for subsequent use on ice;
4) after mixed liquor A and mixed liquid B equal-volume being mixed, be seeded to 24 orifice plates immediately, every pore volume is 500 μ L; 37 DEG C of insulation 15min, treat that it is frozen into soft gel;
5) above soft gel, add perfect medium 1mL, be placed in cell culture incubator, culture condition is 37 DEG C, 5.0%CO
2, saturated humidity, every 2 ~ 4 days replaced medium are once;
6) observe the formational situation of tumor stem cell microballoon every day, take pictures; Be cultured to 9th ~ 11 days, microsphere diameter can reach 50 ~ 100 μm, as shown in Figure 1.
embodiment 2
1) by tryptic digestion MCF-7 clone, obtain the MCF-7 cell of unicellular, add in perfect medium (being added with the DMEM in high glucose substratum of 10v/v% foetal calf serum, 150U/ml penicillin and 150mg/ml Streptomycin sulphate), make breast cancer cell suspension, with perfect medium (being added with the DMEM in high glucose substratum of 15v/v% foetal calf serum, 150U/ml penicillin and 150mg/ml Streptomycin sulphate), cell density is adjusted to 1 × 10
3cell/mL;
2) salmon zymoplasm (0.1U/ μ L) 5 μ L are mixed with 250 μ L cell suspensions, obtain mixed liquor A, be placed in for subsequent use on ice;
3) with perfect medium, salmon Fibrinogen is diluted to 1.5mg/mL, obtains mixed liquid B, be placed in for subsequent use on ice;
4) after mixed liquor A and mixed liquid B equal-volume being mixed, be seeded to 24 orifice plates immediately, every pore volume is 500 μ L; 36 DEG C of insulation 18min, treat that it is frozen into soft gel;
5) above soft gel, add perfect medium 1mL, be placed in cell culture incubator, culture condition is 37.5 DEG C, 5.1%CO
2, saturated humidity, every 2 ~ 4 days replaced medium are once;
6) observe the formational situation of tumor stem cell microballoon every day, take pictures; Be cultured to the 11st day, microsphere diameter can reach 60 ~ 100 μm.
embodiment 3
1) by tryptic digestion MCF-7 clone, obtain the MCF-7 cell of unicellular, add in perfect medium (being added with the DMEM in high glucose substratum of 15v/v% foetal calf serum, 100U/ml penicillin and 100mg/ml Streptomycin sulphate), make breast cancer cell suspension, with perfect medium (being added with the DMEM in high glucose substratum of 12v/v% foetal calf serum, 100U/ml penicillin and 100mg/ml Streptomycin sulphate), cell density is adjusted to 1 × 10
5cell/mL;
2) salmon zymoplasm (0.1U/ μ L) 20 μ L are mixed with 500 μ L cell suspensions, obtain mixed liquor A, be placed in for subsequent use on ice;
3) with perfect medium, salmon Fibrinogen is diluted to 3mg/mL, obtains mixed liquid B, be placed in for subsequent use on ice;
4) after mixed liquor A and mixed liquid B equal-volume being mixed, be seeded to 24 orifice plates immediately, every pore volume is 500 μ L; 38 DEG C of insulation 15min, treat that it is frozen into soft gel;
5) above soft gel, add perfect medium 1mL, be placed in cell culture incubator, culture condition is 37 DEG C, 4.9%CO
2, saturated humidity, every 2 ~ 4 days replaced medium are once;
6) observe the formational situation of tumor stem cell microballoon every day, take pictures; Be cultured to the 9th day, microsphere diameter can reach 50 ~ 80 μm.
testing index:
(1) cellular form is observed: the formational situation observing tumour microballoon every day, takes pictures.9th ~ 11 days, count the microballoon formed under the microscope, its rate of formation was about 4 ~ 10%.
(2) detection of stem cell labeling thing: collecting cell, adopts immunofluorescence technique to carry out the detection of marker, identifies whether it has CD44
+/ CD24
-/lowphenotype.
Example 1 to be performed is cultured to the 10th day, by being mixed with the gel inoculation of cell on the cover slip, fix 30min with the paraformaldehyde of 4%, the penetrating 10min of TritonX-100 of 0.5%, 30min closed by 10% bovine serum, add CD44 primary antibodie (CD44 antigen isoform 4 antibody, Pierce), CD24 primary antibodie (CD24 Antibody, Pierce) overnight incubation respectively, rinsing, then two anti-(goat anti-rabbit igg, FITC mark, sigma of FITC mark are added respectively; Donkey anti-mouse IgG, FITC mark, invitrogen) hatch 45min, finally to dye 10min with DAPI again, drip upper mountant, take pictures at fluorescence microscopy Microscopic observation.
As shown in Figure 2, left figure is after the CD44 antibody incubation that FITC marks, and under fluorescent microscope, cell presents bright yellow-green fluorescence, is indicated as the CD44 positive for result; The CD24 antibody that right figure FITC marks fails signal to be detected, only sees and is indicated as CD24 by the blue cell core that DAPI dyes
-/low.This result meets the phenotype CD44 of breast cancer tumour stem cell
+/ CD24
-/low.The mrna expression amount of ESA, CD44, CD24 is detected: collect the cell that embodiment 1 is cultured to the 10th day by real-time fluorescence quantitative PCR, the total serum IgE of extracting cell, gets a certain amount of RNA reverse transcription and becomes cDNA, preparation PCR system, upper machine testing, compares CT method by δ δ and carries out data processing.
Same aforesaid method, detects the mrna expression amount of ESA, CD44, CD24 of the MCF-7 cell before cultivating screening.
As shown in Figure 3, the tumor stem cell of acquisition is compared with before screening, and the expression of itself ESA and CD44 gene all has remarkable rising, and CD24 there is no considerable change, meets breast cancer tumour stem cell ESA for result
+/ CD44
+/ CD24
-/lowphenotypic characteristic.
(3) expression amount of CD44, CD24 albumen is detected by Western-blot: collect the cell that embodiment 1 is cultured to the 10th day, extract proteins, get after a certain amount of total protein carries out SDS-PAGE electrophoretic separation, electrotransfer is on nitrocellulose membrane, skim-milk is closed, add primary antibodie (CD44 antigen isoform 4 antibody, Pierce respectively; CD24 Antibody, Pierce) hatch, two anti-(goat anti-rabbit iggs (H+L), HRP marks, Pierce and sheep anti-Mouse IgM, HRP mark, Pierce) hatch, luminous, development, takes pictures, software analysis optical density value.
Same aforesaid method, detects the expression amount of CD44, CD24 albumen of the MCF-7 cell before cultivating screening.
Result as shown in Figure 4, Western-blot detected result shows, through the cell that this three-dimensional culture method filters out, and expression amount remarkable increase compared with before screening of CD44 albumen, expression amount remarkable reduction compared with before screening of CD24 albumen, result meets the phenotype CD44 of breast cancer tumour stem cell
+/ CD24
-/low.
Claims (5)
1. screen a three-dimensional culture method for breast carcinoma stem cell, comprise the steps:
1) obtain the human breast cancer cell of unicellular, add in perfect medium, make cell suspension, cell density is adjusted to 1 × 10
4~ 1 × 10
5individual/mL; Described human breast cancer cell is MCF-7;
2) salmon zymoplasm is added cell suspension, the concentration making zymoplasm is 1 ~ 4U/mL, and mix, obtain mixed liquor A, ice bath is for subsequent use;
3) with perfect medium, salmon procollagen is diluted to 1.5 ~ 3mg/mL, obtain mixed liquid B, ice bath is for subsequent use;
4) mixed liquor A and mixed liquid B equal-volume are mixed, obtain mixed solution C, be seeded to Tissue Culture Plate, be incubated at 36 ~ 38 DEG C, make it be frozen into soft gel;
5) above soft gel, add the perfect medium that volume is mixed solution C1 ~ 2 times, be placed in cell culture incubator and cultivate, every 2 ~ 4 days replaced medium once;
6) be cultured to 9 ~ 11 days, namely obtain breast carcinoma stem cell microballoon.
2. method according to claim 1, is characterized in that: described perfect medium is the DMEM in high glucose substratum being added with 10 ~ 15v/v% foetal calf serum, 80 ~ 150U/ml penicillin and 80 ~ 150mg/ml Streptomycin sulphate.
3. method according to claim 1, is characterized in that: step 2) in, in mixed liquor A, the concentration of salmon zymoplasm is 4U/mL.
4. method according to claim 1, is characterized in that: in step 3), and in mixed liquid B, the fibrinogenic concentration of salmon is 2mg/mL.
5. method according to claim 1, is characterized in that: in step 5), and culture condition is: 37.0 ~ 37.5 DEG C, 4.9 ~ 5.1%CO
2, saturated humidity.
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| CN105087494A (en) * | 2015-09-11 | 2015-11-25 | 中国人民解放军第四军医大学 | Culture method of breast cancer stem cells |
| CN108130314B (en) * | 2018-01-24 | 2022-05-17 | 郑州大学第一附属医院 | Monoclonal cell culture method |
| CN110396501B (en) * | 2019-08-01 | 2020-10-30 | 江苏省人民医院（南京医科大学第一附属医院） | Three-dimensional spheroid culture method for maintaining dryness of breast cancer stem cells in vitro |
| CN114525251A (en) * | 2022-02-21 | 2022-05-24 | 广东粤微食用菌技术有限公司 | In-vitro rapid culture method of 3D breast cancer cells |
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| CN102395673A (en) * | 2009-03-20 | 2012-03-28 | 首尔大学校产学协力团 | Isolating method for umbilical cord blood-derived pluripotent stem cells expressing |
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